The purpose of this tutorial is to provide a step-by-step guide to
isolate nuclei from flash-frozen tissue for single nucleus RNA
sequencing. Nuclei Isolation is the first step in many novel techniques
including snNRAseq, single-nucleus assay for transposase-accessible
chromatin sequencing, epigenomic analysis. Although based off of the 10X
Genetics Chromium Nuclei Isolation Protocol, this tutorial is
specifically for the use of kidney tissue for snRNAseq and includes
alterations to maximize yield and quality. This tutorial will also
include a guide on how to assess and grade the quality of the isolated
nuclei as well as next steps after the initial isolation.
Edited Visual Diagram of Protocol, with inserted notes for easy
reading:
3 Protocol:
The protocol includes four main steps: buffer set up, lysis, debris
removal, and resuspension.
3.1 Buffer Set-Up
Table of Buffers:
Buffer Name
Component 1
Component 2
Compound 3
1X PBS
10 mL of 10X PBS
90 mL DI water
N/A
10% BSA
10 g BSA powder (4°C)
100 mL DI water
N/A
Lysis Buffer
Reducing Agent B (-20°C)
Surfactant A (4°C)
Lysis Reagent (4°C)
Debris Buffer
Reducing Agent B (-20°C)
Debris Removal Reagent (4°C)
N/A
Wash and Resuspension Buffer
1X PBS
10% BSA (4°C)
Rnase Inhbitor (-20°C)
1. Start with the Wash and Resuspension
Buffer, which is composed of the 1X PBS and 10% BSA solutions that are
not included in the kit. Generally, make a stock of 100 mL of each and
keep chilled at 4 degrees Celcius. Refer to the table above and the
instructions for components and amounts.
1X PBS: Dilute 10 mL into 90 mL of Ultra-Pure
Water. Invert to mix thoroughly.
10% BSA: 10 grams of powder BSA to 100 mL of
Ultra-Pure Water. Mix gently by swirling to prevent excess frothing.
2. Next, retrieve all components of the 10X
Nuclei Isolation Kit, which should include the Lysis Reagent, Debris
Removal Reagent, Reducing Agent B, Surfactant A, and RNAse Inhibitor.
Also retrieve the Sample Dissociation Tube from the room temperature
Consumables portion.
3. Place everything except for the Reducing
Agent B on ice and begin to prepare the buffers. After Reducing Agent B
thaws and everything is briefly vortexed, begin preparing the
buffers.
4. Per reaction, the Lysis Buffer requires
550 µl Lysis reagent, 0.55 µl Reducing Agent B, and 5.5 µl Surfactant A.
Per reaction, the Debris Removal Buffer requires 550 µl Debris Removal
Reagent and 0.55 µl of the Reducing Agent B. Per reaction, the Wash and
Resuspension Buffer requires 2,887.5 µl 1X PBS, 330 µl 10% BSA, and 82.5
µl of RNase Inhibitor for sequencing.
5. Keep all completed buffers labeled and on
ice. Pre-chill the centrifuge to 4 degrees C.
3.2 Main Procedure
1. Retrieve flash frozen half kidney from the
-80 degrees storage. While frozen, use tweezers or scissors to remove
the kideny and place it in a pre-chilled Sample Dissociation Tube.
2. Add 200 µl of the Lysis Buffer and wait
for the kidney to mostly thaw. Using sterile scissors, chop up the
kidney into small pieces, closing and opening scissors at least 50
times.
3. Then using the green pestle provided in
the room-temperature pack, grind up the tissue, pressing down and
twisting at least 20 times or until the solution is smooth with no
visible chunks.
4. Next, add 300 µl of the Lysis buffer and
pipette up and down, making sure that there are no chunks that block the
pipette tip. Incubate the solution on ice for 8 minutes, starting the
timer after completing the grinding step.
5. Next, pipette the dissociated tissue
solution onto the Nuclei Isolation Column in the collection Tube (both
included in room temperature Consummables) and spin in the centrifuge
for 16,000 rcf, 20 sec, at 4 degrees C. Make sure that this cycle is
evenly balanced in the centrifuge, because of the high speed.
6. After the cycle ends, discard the column
and vortex the flow-through for 10 seconds to resuspend the nuclei. Then
spin in the centrifuge at 700 rcf, 3 min, 4 degrees C. Make sure that
when placing the Nuclei Isolation Column, position it within the
centrifuge so that the hinge of the lid points out, the front tab facing
in. This will make it easier to find the pellet and avoid losing
nuclei.
7. Next, remove the supernatent, being
careful to avoid the pellet and angling the pipette tip away from the
hinge side of the column.
8. Resuspend the pellet with 500 µl of the
Debris Removal Buffer. Spin at 700 rcf for 10 minutes at 4 degrees C.
While waiting, mix the final Resuspension Buffer, which should be 0.04%
BSA in PBS.
9. Remove the supernatant again and resuspend
in 1 mL of the Wash and Resuspension Buffer. Spin at 500 rcf for 5
minutes, 4 degrees C. Remove supernatant, resuspend again in 1 mL of
Wash and Resuspension Buffer. Spin again at 500 rcf for 5 minutes, 4
degrees C.
10. Finally, resuspend the. nuclei pellet in
500 µl of the Final Wash and Resuspension Buffer (0.04% BSA). Vortex the
solution for 3 seconds and let it sit undisturbed for 15 minutes.
Now, depending on QC or following uses, use the concentrated
solution.
3.3 Nuclei Count with
Light Microscope:
With the nuclei solution, add 2 µl to 18 µl of Trypan Blue, pipetting
up and down into the TB at least 10 times to mix thoroughly. Note that
while Trypan Blue does not aggregate in whole cells, the compound does
aggregate within the exposed nuclei, which is what we will be checking
for. Add 10 µl to one of the wells on a hemocytometer, making sure the
solution fills the space. Observe on a light microscope, counting to get
an approximation and assessment of nuclei quality.
From this procedure, the average expected nuclei concentration count
is: 500 nuclei per microliter This is approximately 20 to 40 nuclei per
quadrant in the hemocytometer.
3.4 Nuclei Quality
Check:
Priority Check:
No clumps, with an even distribution
Quality:
A grade Nuclei: Good, circular shape with no blebbing, halo of Trypan
Blue around the edges that suggest an undisturbed nuclear envelope. No
aggregation.
B grade Nuclei: Slightly irregular shapes, still has the halo but
less bright or more uneven
C grade Nuclei: Losing more of normal shape, small blebbing, paler
color
D/F grade Nuclei: Complete loss of structure, no blue halo,
aggregates
Example of a graded sample:
Example of grading with reasoning. Note: this is an image of an
aggregate that was included because of a wide variety of quality, but as
a general rule aggregates indicate a low quality overall sample.
Example of a sample viewed at 10x and 40x, grading the individual
nuclei quality is easier at 40x but it is recommended to conduct overall
sample quality checks at 10x magnification.